Project Summary Spinal cord injury (SCI) affects approximately 300,000 Americans resulting in devastating neurological and physical limitations. SCI in the chronic phase is complicated by muscle spasms, which are to a large degree caused by hyperactivation of the serotonin receptor 2C (5HT2C) caudal to the injury. Through a combination of alternative splicing and editing, the G-protein coupled receptor generates at least 25 isoforms with different regulatory properties: one intracellular truncated receptor 5HT2C_tr, one non-edited full-length receptor 5HT2C_Fl_INI and 23 full length edited receptors 5HT2C_FL_ed. The ratio of these isoforms wherein the 5HT2C_FL shows constitutive activity plays a major part in the maladaptation after injury. SCI leads to a loss of editing activity of ADAR2 (adenosine deaminase acting on RNA), which results in a downregulation of 5HT2C_FL_ed isoforms, relative to the 5HT2C_FL_INI. Extending our joint published work (Zhang et al., 2016, EMBO Mol. Med)1, we concentrated on the novel 5HT2C_tr isoform that sequesters the 5HT2C_FL intracellularly. We found an increase of the 5HT2C_tr isoform after SCI, possibly as an adaptation to prevent the constitutively active 5HT2C_FL_INI from hyperactivity. To intervene with the isoform ratios, we developed a series of oligonucleotides that either increase or decrease the 5HT2C_tr /5HT2C_FL_INI ratio, as well as an antiserum that is specific for the 5HT2C_tr protein. Using these tools, we will test the hypothesis that SCI leads to an increase in both constitutively active 5HT2C_FL_INI and its downregulating 5HT2C_tr isoform. This change deregulates spinal cord neuronal activity leading to spasms, likely due to insufficient 5HT2C_tr isoform to stop the 5HT2C_FL_INI activity. We will thus further test whether these spasms can be mitigated using our developed oligonucleotides that promote 5HT2C_tr. Using an established S2 spinal cord transection rat model, we will map the spatial and temporal changes in receptor isoforms and test the influence of splicing-changing oligonucleotides on the development of tail spasms. The oligonucleotides interfere with the serotonin system by changing the surface localization of constitutive active 5HT2C_FL, which is a novel approach compared to ligand- based drugs activating surface receptors. Using this innovative approach targeting the pre-mRNA, these studies are significant with great potential to treat spasticity, which is a major co-morbidity of SCI.